Metastatic cancer is difficult to treat and is often incurable. Characterizing pro-metastatic signaling mechanisms is therefore crucial for the identification of new therapeutic targets for metastasis and the improvement of clinical prognosis through the development of novel molecular therapies. Dysregulated protein kinase signaling is essential in promoting cancer and metastasis progression. One of the most dysregulated signaling pathways in human cancer, aberrantly activated in up to 30% of all human cancers, is the Ras-Raf- MAPK/ERK pathway. Understanding resulting cellular changes from increased MAPK signaling is therefore crucial in determining cancer cell signaling required for tumorigenesis and metastasis. The p90 ribosomal S6 kinase (RSK) family of kinases are substrates of ERK and can mediate many of the biological functions of the Ras/ERK MAP kinase pathway through their activity as serine/threonine kinases. We recently determined that RSK isoform 2, RSK2, promotes cancer cell invasion, migration, and resistance to anoikis in vitro. In addition, we identified RSK2 as a critical regulator of head and neck cancer metastasis in vivo. We recently identified stathmin, a microtubule destabilizing protein, as a potentially novel RSK2 substrate via a phospho-proteomics study. We observed that RNAi mediated knockdown of RSK2 attenuates the phosphorylation of stathmin on a key regulatory residue, Serine 16. We previously demonstrated that RSK2 regulates stabilization of actin filaments in head and neck cancer cells, which suggests that RSK2 may play a larger role in cell cytoskeletal regulation. In this proposal, we will employ multiple approaches to test our hypothesis that RSK2 phosphorylates stathmin to promote a pro-metastatic phenotype in cancer. The specific aims of the proposed research are 1) to explore whether RSK2 phosphorylates and regulates the activity of stathmin to modulate microtubule polymerization at the leading edge of the cell and 2) to determine whether RSK2 signals through phosphorylation of stathmin to promote cancer cell invasion and tumor metastasis. In the long term, we seek to comprehensively characterize RSK2 and downstream signaling effectors, which will be critical to providing a greater understanding of the signaling mechanisms underlying metastatic progression.